Genealogical Data in Population Medical Genetics: Field Guidelines

Genealogical data in population medical genetics: Field guidelines

Fernando A. Poletta1,2, Ieda M. Orioli1,3 and Eduardo E. Castilla1,2 1Estudio Colaborativo Latinoamericano de Malformaciones Congénitas, Instituto Nacional de Genética Médica Populacional, Rio de Janeiro, RJ, Brazil. 2Latin American Collaborative Study of Congenital Malformations, Center for Medical Education and Clinical Research, Buenos Aires, Argentina. 3Departamento de Genética, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

Abstract This is a guide for fieldwork in Population Medical Genetics research projects. Data collection, handling, and analysis from large pedigrees require the use of specific tools and methods not widely familiar to human geneticists, unfortunately leading to ineffective graphic pedigrees. Initially, the objective of the pedigree must be decided, and the available information sources need to be identified and validated. Data collection and recording by the tabulated method is advocated, and the involved techniques are presented. Genealogical and personal information are the two main components of pedigree data. While the latter is unique to each investigation project, the former is solely represented by gametic links between persons. The triad of a given pedigree member and its two parents constitutes the building unit of a genealogy. Likewise, three ID numbers representing those three elements of the triad is the record field required for any pedigree analysis. Pedigree construction, as well as pedigree and population data analysis, varies according to the pre-established objectives, the existing information, and the available resources. Keywords: medical genetics, population medical genetics, geographic clusters, isolates, rare diseases.

Introduction named Aicuña is offered as an example throughout this pa- When the medical genetics of very large families, per in italic text. Further information about Aicuña has been demes, or populations need to be studied, the field of Popula- published elsewhere (Castilla and Adams, 1990, 1996; tion Medical Genetics is utilized. Population Medical Genet- Bailliet et al., 2001). ics is a medical genetics subspecialty which main concern is to understand the causes of prevalence as well as the mainte- Field Guidelines for Collection, Handling, and nance of unusual diseases in populations, generally, rural, Analysis of Genealogica Data isolated, and small, as conceptually defined and discussed elsewhere (Castilla and Orioli, unpublished data). Pedigree objectives Data collection, handling, and analysis of large pedi- Before you start interviewing people and recording a grees require the use of specific tools and methods not family history, reflect until you have a precise idea of why widely known to human geneticists. Unfortunately, the are you going to do it as well as an approximate notion of graphic pedigree is the most popular tool in medical genet- the expected workload. ics in spite of its severe limitations in terms of family data The objective of a pedigree could be the recording of recording, storage, and analysis. a breeding structure, family or population in which a This paper is intended to serve as a guide for field- proven or suspected mutant gene is segregating or some de- work in population medical genetics research projects. To gree of endogamy might be present. While the former in facilitate the understanding of the recommendations pro- most cases is applied to families in which the type of inheri- vided and their underlying concepts, a real population tance is to be proven and/or individuals at risk are to be identified, the latter is usually the case in populations with a Send corresponce to Fernando A. Poletta. Estudio Colaborativo higher than expected frequency of some deleterious pheno- Latinoamericano de Malformaciones Congénitas, Centro de Edu- type. cación Médica e Investigaciones Clínicas, Dirección de Investi- gación. Galván 4102, Buenos Aires (1431), Argentina. E- mail: Even though there is not a clear-cut distinction be- [email protected]. tween a family and a population, some idea of the number 172 Poletta et al. of individuals (pedigree members) to be recorded is needed Personal data in advance, or at least the order of magnitude. For instance, The second block contains the individual’s personal a large family seldom reaches one hundred persons, even information, such as, for instance, gender, full name, date including deaths, while the genealogical size of a popula- and place of birth, marriage and death, phenotype, etc. tion will usually be greater than one thousand and could Likewise, its length is unlimited because and each re- even be as high as ten thousand. searcher can structure it as preferred, and links with other When dealing with a population, be sure you actually databases is both possible and desirable. need a pedigree because, depending upon your objective, performing a census or a register will be a better approach Tabulated data recording in many situations. The problem of graphic pedigree recording Initially, briefly describe the population to be studied, Graphic pedigrees are very limited from all points of its size, and relevant characteristics. Also describe the geo- view. Their capacity for individuals is limited to less than graphic area where the population is located with precise one hundred, while genealogical or historical populations limits, preferably within an administrative unit if it can be in most isolates are in the order of tens of thousands. The identified (municipality, region, state, etc.) in order to ob- gametic links among individuals very soon become an tain background information when required. incomprehensive set of interlocking and overlapping lines. Nature of collected information The allowed personal information is limited to gender, indicated by squares or circles, plus less than ten similar data As any living organism, families are as dynamic as points, such as live-dead status, birth-death years, a couple the individuals composing them. Therefore, their informa- of genomic indicators, and nothing else. Furthermore, the tion is neither complete, nor conclusive, and all collected most limited value of pictorial genealogies is not related data are to be taken as provisional and conditional. primarily to its use as a database but rather to its ineffi- Information components: Informer and individual ciency for use in data analysis. As a matter of fact, the only utility of pictorial representation of family trees is to dis- In practical terms, any pedigree must identify the in- play a family genetic structure and/or disease distribution former and the individual or pedigree member. For in- that has already been studied and proven by other methods. stance, a person numbered 001_017 is understood as Even this depiction is bound to appropriate signs and no- individual # 017 provided by informer # 001. Because dif- menclature. Graphic symbols and keys follow pre- ferent collected pedigrees of a given family or population established standards provided by specialized journals or do overlap or even repeat the same information, a single in- societies as summarized by Bennett et al. (2008) for the use dividual can have more than one number, and the hypothet- of genetic counselors. ical person 001_017 may simultaneously be person 023_133. Data collection form These provisional numbers primarily serve to orga- The sample page shown in Table 1 illustrates the pro- nize field notes. Regardless of the numbering, the identity cess of data recording during the inquiry process. General of a given individual is based on its genealogical links. Dif- information at the top of the page includes the identification ferent numbers with the same parents and descendants can of the Informer and the Operator, both by means of a code only refer to the same individual, and any information at number, plus the date and place of data collection. The form odds with this assumption must be investigated and cor- contains the following two distinct parts: Genealogy, com- rected. prised by a triad of ID numbers for the person in a given row Once the identity of individuals within the population and the father and mother; and Personal Information, is established, the next step is to resolve any differences in which here, solely for demonstration, includes gender, life the information contained on separate forms and finally to or death status, year of birth, death, and emigration, and an assign an identification number to the individual in ques- amputated field for first name, indicating the continuity of tion. the form following the right border. Some usual and also Each individual record contains two distinct blocks of special situations are illustrated, namely the following: information concerning genealogical and personal data. Numbering of individuals within the pedigree is abso- lutely free and up to the operator decision. The only rule is Genealogical data not to use the same number for more than one pedigree The first block is comprised of a unique number iden- member, since each individual number is an identity num- tifying the nuclear family unit or basic triad, namely, the in- ber. Conversely, a skipped or unused number creates no dividual and his/her father and mother. Each number problem in pedigree analysis. cannot be given to more than one individual. Nevertheless, Some pieces of information require specific working consecutivity, or sequentiality, is not necessary, and num- definitions. For instance, for migration information, a local bering can be discontinuous. territory needs definition first, as in, for instance, a munici- Genealogical data 173

Table 1 - Tabulated genealogy: Data collection form.

Informer ID: ### Operator ID: ### Date/S:--/--/-- Place/S: Pedigree ID: ### Personal information Individ Father Mother G L BIR-YR DTH-YR Name AFF 001 004 003 F 2 002 004 003 M 2 003 005 006 F 1 004 007 008 M 1 005 014 009 M 1 006 999 999 F 1 007 017 016 F 1 008 013 012 M 1 009 013 012 F 1 010 013 012 F 1 011 013 012 M 1 012 999 999 F 1 013 024 023 M 1 014 999 999 M 1 015 017 016 M 1 016 021 020 F 1 017 999 999 M 1 018 021 020 M 1 019 021 020 F 1 020 024 023 F 1 021 999 999 M 1 022 024 023 F 1 023 999 999 F 1 024 999 999 M 1

G: Gender: M male, F female, I indetermined, blank: unkown. L: Live or death status; BIR-YR: Year of birth; DTH-YR: Year of death (if dead); AFF: Affected status 1 (No); 2 (Yes). pality. Moving into a region, if born outside, or out of a re- In the absence of a specially designed data-recording gion, if born inside, define patterns of migration (immigra- form, a simple sheet of squared paper can very well do the tion or emigration) constituting important components of job. Likewise, a simple spreadsheet application can be eas- the genetic structure and dynamics of a given population. ily formatted to serve as a data-recording form if direct data The correspondence between tabulated and graphical keying is thought to be suitable for the planned fieldwork genealogy is shown in Table 1 and Figure 1. In the example process. presented here, the number 001 individual seems to be the index case because their ancestors and sibs are recorded. Information sources: Oral and written Individual # 002 is also affected and is the brother of In general terms, the reach of well-informed collabo- the index case because both have the same parents (# 004 rative oral informers reaches back to ancestors born in the and # 003). mid 19th century. Considering that we are working at the Individuals # 005 and # 006 are the maternal grand- beginning of the 21st century, that a current ancient in- parents, while # 007 and # 008 are the paternal grandpar- former could had be born in the 1930s, and that generation ents. length is estimated to be approximately 30 or 35 years, the Individual # 006 was fathered by an unknown per- informer parents were born around the year 1900, grand- son; however, it is known that these parents were mem- parents around 1870, and great-grandparents about 1840. bers of the isolated community and were therefore num- Any information previous to that time limit must be bered 999. restricted to written informers. The relevance of this distinction is that for inbreeding Information on extended genealogy is limited to a calculations, while members of the 000 sibship are assumed small number of informants who have a certain vocation for to be outbred, those of the 999 group will have the average family history and moreover enjoy recounting information expected inbreeding coefficient of his/her generation. that they received from their parents. Nevertheless, an in- 174 Poletta et al.

given population pedigree must be understood as a minimal estimate of reality. This issue will be further covered in section “Illegitimacy, Confidentiality, and other Ethical issues”.

Oral sources For family groups, the best informer is a knowledge- able member of that group. For more remote genealogical data, usually related to the population founders and early generations, the best informers could be just one or a few well-informed persons, usually well known by all community members. In isolated populations, the reliability of the informants is reinforced by endogamy, which reduces the number of ancestors (loss of predecessors), as well as by a feeling that could be called the “genealogical conscience”.

Evoked persons are not fully dead Even though the knowledge of genetic links among members of past generations may be relatively limited, a general knowledge of them is shared by all. There is a particular awareness of and sensitivity towards the dead that may not be exclusive to just one population; rather, this Figure 1 - Graphical pedigree of the tabulated genealogy shown in sentiment so eloquently evoked by Gabriel García Márquez Table 1. for many of the characters in Cien Años de Soledad (1973), may be common to all rural communities in Latin America. In Aicuña, this sentiment results in the impression that no terest in history may not be consonant with an interest in ge- one ever really dies; many decades after physiological nealogy. Usually, a small number of “amateur genealo- death, an individual lives on in legends and anecdotes such gists” is sufficient to maintain oral traditions and to prick as “the stone of Don Rosario”, “the spring of the Olivas” or the “genealogical conscience” of the community at large. “the footpath of Severino”. Conscious or not, genealogical interest cannot be Each place named after a deceased person usually co- solely based on a pure sense of belonging, or even of eter- mes with a story that revives him/her, as well as the lifestyle nity, because a family spreads over time, making a predom- and conditions at that time, helping the whole process of inant last name almost immortal, as in the case of the memory recall needed during the building of a large pedi- Buendias of Macondo, the Terra-Cambarás of Santa Fe, or gree. Furthermore, this preeminence of the dead in the col- the Ormeños of Aicuña (Castilla and Adams, 1996). lective consciousness of the community greatly facilitates Genealogical interest could be directly linked to land the task of oral informers. and property rights, which, whether effective or not from the legal standpoint, provides the members of a genealogy Close and remote informants with a perhaps unperceived but attractive sense of aristoc- Obviously, people know more of their family group racy. This was the reason for the genealogy of the pioneer than others. Different degrees of extension can be estab- generations in New England (Greven, 1972), the Petrorca lished between the nuclear family, or parental triad, and the Valley in Chile (McCaa, 1983), and also in Aicuña (Castilla whole kinship composing the whole community at the level and Adams, 1990). The influence of land property on the of household, grandparents, descendants, etc. The recom- population genetic structure has been demonstrated be- mended strategy to generate a large pedigree will vary from cause family branches with more land tended to emigrate case to case, largely depending upon the research objec- less, therefore increasing the mean inbreeding coefficient tives and population size and distribution. across generations in the 19th and 20th centuries (Castilla and Adams, 1990). Current and ancient informants Natural, or illegitimate, offspring might be hided or The majority of informers are able to provide reliable even masked by attributing a wrong paternity. The latter is information on their own generation and the subsequent particular of out of the wedlock conceptions. Since the im- one, namely their own offspring. Youth in general demon- pact of this type of errors is difficult to validate, even if sus- strate very limited interest in their ancestors, and elders do pected, coefficients of relationships and inbreeding for a not know much about infants and children. Genealogical data 175

Aicuña (example 1): At the end of a week of inter- Although Julián and Luisa were one of the best views, in spite of frequent digressions, two of the infor- sources of information in Aicuña, there were at least 10 mants had provided both genealogical and demographic others with similar characteristics. While they were being information on a total of 795 present and past members of interviewed, the village nurse listened because the inter- the community. Don Julián’s parents were born in 1863, views took place in a small clinic in the village that was un- and his great-grandparents, who were born between 1790 der her responsibility. She herself, 35 years old at the time, and 1801, were the offspring of persons censed in Aicuña in had a special interest in the contemporary generations of 1810 and signatories of the legal action creating the two es- Aicuña and was able to provide information on 1036 indi- tancias of Aicuña and Tambillos in 1833. Thus, it is hardly viduals. When the data were updated, some 15 years later, surprising that such an informant was able to provide reli- after the death of Julián and Luisa, the nurse had become able data from the beginning of the 19th century on, espe- the best informant in the village for the past generations, cially considering that the inhabitants of small having acquired a considerable amount of information in consanguineous communities, with little immigration, usu- the intervening years from the elders in the village. Inter- ally possess good information on their ancestors. estingly, a new nurse in the village had taken on the role of being the informant for the contemporary population. Uni and multi personal sources Thus, a new generation of informants had arisen in Aicuña The sources of oral information include isolated indi- to guarantee the continuity of the oral tradition, which was viduals or groups of two or three persons, whether related a responsibility, according to standards of the community, or not and residing in town or not. Within a group of two or which, although not written, were no less rigid that those three informers, one usually is the primary source of infor- that were. mation, while the others corroborate the information or pro- Illegitimacy, confidentiality and other ethical issues vide additional information on a particular member of the Everyone is capable of providing information on their pedigree. immediate family, and no one can provide better informa- In all other cases, groups of two or three informers are tion on a given paternity than the mother of the child, pro- preferable to single individuals because discussion of a cer- vided she is willing to do so. Further information on these tain individual or ancestor frequently serves to stimulate issues is provided below in Section “Ethics for field work”. the memory, often with the result of giving more credibility Willingness to the data. The reluctance to cooperate in providing genealogical Aicuña (example 2): The 49 informants utilized in- information may have several underlying reasons. Usually cluded single individuals such as informant #008, groups of they are not voiced or may be unclear for the person ap- two or three persons, such as informants #003, Don Julían proached. However, that unwillingness should be respected and Doña Luisa. These individuals were located in the vil- without putting any pressure on the individual, not only be- lage of Aicuña (e.g., #003, and #008) in the city of cause of ethical principles but also in order not to enter in- Chilecito, such as informer #012, or even 1,300 km away in correct gametic links into the pedigree in question. Buenos Aires, such as informer #1. This last informant was Because a 0000, or even better, a 9999, parenthood is the first one interviewed because he lived in the city where largely preferable to a mistaken number given, thanks the population survey was designed. He served as a “test” should be expressed for the refusal, even when a final, more for the data forms used in the field before the final versions vague question could close the conversation, as described were being printed. Informer #001, aged 62 when the inter- in the next section below. views took place, was able to provide genealogical and demographic information concerning 182 members of his llegitimacy immediate family and descendants. While all biological offspring are natural , irrespec- The most prolific oral source of information was most tive of their legal or religious status; illegitimate refers to likely group #003, which was already mentioned. Don being illegal, illicit, or even criminal, in reference to a given Julián was 81 years old when he was interviewed, and set of civil or religious rules that are valid for a given cul- Doña Luisa was 72. Julián and Luisa were much more in- ture at a given time and place. Natural should be preferred formative as a couple than on their own. When talking to illegitimate under the assumption that the real meaning is about a branch of the family tree that was not very well natural and illegitimate. Out of wedlock, meaning outside known, in general concerning families that had emigrated, of matrimony, is another way to designate a conception oc- they would typically begin to digress, telling stories that curred during a valid matrimony but was seeded by a per- eventually sparked their memories, allowing them to re- son not the formal spouse. trieve dates and genealogical information. They themselves It is wise to investigate the terms and their real mean- would say that it was preferable to have each other present ings employed in the community. Likewise, the general at- “para no dejarlo mentir” (to stop them from lying). titude toward parenthood should be understood. So-called 176 Poletta et al. illegitimate paternities are not necessarily sinful or shame- Ecclesiastic log books ful. In Latin America, church records refer exclusively to Nevertheless, illegal paternities are rarely recorded in the Roman Catholic Church, primarily if working with log th written sources of information such as vital statistics, books from the 19 century and earlier. These books are church records, or legal documents. very similar in different countries and regions, as well as in The effort to be invested to define a given gametic different languages, such as Spanish, Portuguese, or others. link largely depends upon its genealogical value, measured Parish records have considerable genealogical and as the contribution of the unknown person to the genome of demographic interest because they contain the following subsequent generations or even to the genetic pool of the four different categories of information: baptisms, mar- current demographic population. riages, confirmations and deaths. Three of them reflect vital events (birth, marriage, and death), while one (confirma- Usually, crucial genetic inputs to the genealogy oc- tion) could be a proxy for a population census. curred several generations back, and it is very unlikely to Church books are usually well kept and stored in depend upon the unrevealed paternity of a recently born good conditions although some of them may be three or child from an adolescent mother. four centuries old. The reading quality is contingent on the Confidentiality preservation of paper and ink and largely depends upon the regional climate; the dryer, the better. Desert regions are Single informers serving as oral sources of informa- ideal for the preservation of books and for any object of ar- tion are generally preferable when information on parent- cheological value. age is concerned because such individuals are more likely Nineteenth and even 18th century handwriting is easy to disclose information on certain paternities that was con- to read after one day of self-taught training, and the few ab- cealed from the rest of the community. This also, naturally, breviations used are easily decipherable – for example Ma. allows this information to remain confidential. for Maria, Fsco. for Fransisco, v. for viuda (widow) or Nevertheless, it is unrealistic to promise full confi- viudo (widower), and so on. dentiality of the collected pedigree information. Even if In general, the quality of the parish registers decline names are concealed, the genealogical links of a given significantly in both organization and detail after the year member within a defined pedigree, finite no matter how 1900, most likely because of the increasing presence of the large it may be, makes it easy to be identified by other pedi- Provincial and State governments and the accompanying gree members. Furthermore, in medical genetics, when the civil records, which relieved the Church of the responsibil- ultimate aim is the prevention of a disease, anonymity ity for recoding vital statistics. Church death recording is could curb the possibility to benefit persons at risk from practically nonexistent because deaths are registered by suffering severe illnesses. governmental civil registry, and burial is no longer limited to the churchyard (camposanto). Other ethical issues Baptism Sometimes, a person refuses to speak about their ancestors because they wish to let their ancestors to rest in Each baptism (Christening) record contains the fol- peace. This could reflect an unwillingness to collaborate lowing information: date of birth, sex, first and last name, with the interrogators instead of a true expression of their first and last names of both parents, or just of the mother in beliefs. The investigator must be aware of this type of atti- the case of a natural birth. tude because if we ignore the reason for a certain reproduc- Records are written in chronological order in several tive pattern in a given family or population, we could also handwritten volumes. be unaware of the possible reasons for concealing informa- In addition, there usually is an alphabetical index, tion. which facilitates any searches for specific members of the pedigree. Written sources Baptism records provide genealogical information for a single unit in two successive generations, a child and Two different procedures can be applied to work with his/her parents. written records: 1. A search for a genealogical path: a string of gametic links following the procedures elaborated in Marriage 6.4.: ascent, descent, sideways. 2. A search for a person:a Each marriage record contains the following informa- genealogy member listed in the alphabetical indices and tion: first and last names and ages of the couple and the first confirmed by genealogical links, in order to complete or and last names of both sets of parents. check personal information such as names, dates, places, These records are also written in chronological order etc. in several handwritten volumes. At a first site visit, identify the available written re- An alphabetical index exists but only for the male cords and the year of the first registration in each log book. partners. Genealogical data 177

Marriage records provide genealogical information erations, a child and his/her parents. As to the latter, each for two linked units in two successive generations, both person is registered with names, gender, and age, allowing spouses and the four parents. This is the most valuable for a population census under these variables, assuming to- church record for genealogical assembly. tal coverage between confirmees and godparents. In the case of consanguineous marriages, the appro- Civil priate ecclesiastical dispensation is recorded with a complete description of the type and level of consanguinity. Civil registry This is hardly surprising because dispensation was required Because civil registers in Latin America were estab- in the Catholic Church until 1917 for marriages between lished at the start of the 20th century, a period easily reached partners as distantly related as third cousins (Royo-Marín, by oral informers active one hundred years later, they have 1958). rarely been used until now. However, those records could be useful to validate personal data for pedigree members, Death such as year and place of birth or death. The nature of the death records varies substantially Analogous to parish records, civil registries also cover over time. First and last names and age are always present, the three basic vital events: birth, marriage, and death. although names of the partners and cause and place of death appear inconsistently. These records are listed chronologi- Elector registry cally in several handwritten volumes and, as for baptism Voter registries are currently available in electronic and marriage records, an alphabetical index also exists. form for many populations. One use for them in human ge- Death registries usually state the cause of death, netics is in the study of last names as phenotypic markers. which is generally useless for medical purposes, except to Several analytical methods have been described for the use differentiate violent from natural causes (“Morte matada e of last names as indicators of ethnicity or breeding structure morte morrida” in Portuguese). Obviously, folkloric or cul- not only in isolated groups but in large populations as well tural cause of death could be useful for other non-medical (Bronberg et al., 2009). specialties. Military service records Confirmation Given that the existence and characteristics of mili- The significance of the confirmation records to the re- tary service records varies among countries and periods, it construction of the demographic and genealogical history is wise to investigate the situation for the population group of the population is somewhat different from the other re- in consideration. cords because the date of confirmation does not correspond In some places, medical examination for military ser- to any important demographic variable, and confirmations vice is the only cross sectional population sampling that ex- are administered to any baptized person of sufficient age. ists after birth, even if it is limited to the male gender in Confirmations are only performed by the bishop, who most Latin American countries. is usually established in a large town far from the locality in Another use of these records is in the historical study which the genealogy originated. A rural region would of anthropometric indexes indicating the nutritional status rarely be visited more than once in the bishop’s lifetime. of a given population through certain time period (Vargas Because these visits occurred so rarely, very few of the in- et al., 2010). habitants would have been confirmed during a previous Personal visit. Therefore, all the inhabitants were confirmed at once, A variety of other documents such as wills, property and the few that had already been confirmed on a previous deeds, and various publications concerning important indi- visit acted as godfathers and mothers. Thus, the confirma- viduals in the family may also exist, which also contain tion records show that a small group of godparents, which valuable information for your research project. included members of the Episcopal cortege, served repeat- edly for the confirmation of one or two hundred persons. In Historical this way, many of the inhabitants of a community and the Considering that a large family or population pedi- surrounding areas were registered at every visit by the gree usually extends over a long time period, and it is in- bishop as confirmees, godparents, or in a few cases as both. serted into a larger human body such as a country or region, In other words, the confirmation records for a given place the local history should be investigated. Cultural or physi- and date can be considered much like a population census. cal catastrophes could influence the breeding pattern with Because the entry for each confirmee provides infor- further genetic effects such as drift. For instance, in the mation on the first and last name, sex, age and parents’ south Atlantic island of Tristan da Cunha, there were two names, these confirmation records contain important gene- unusual events, emigration of over half the population in a alogical as well as demographic information. As to the single year in the mid-19th century, and a major boat disas- former, like baptism, confirmation records provide genea- ter, both of which significantly affected its demographic logical information for a single unit in two successive gen- history (Thompson, 1978). 178 Poletta et al.

Nevertheless, isolated populations could also be un- The most exceptional characteristic of the population affected to the events of the general population. For in- of Aicuña is the existence of a large and complete geneal- stance, no reference was found in Aicuña to the long civil ogy extending over many consecutive generations through war occurred in that Argentine province, La Rioja, during three centuries because the founding of the community. Ta- the 19th century (Castilla and Adams, 1996), or to the ble 2 summarizes the sequence of seven landmark events in Guerra de Canudos in the military garrison, Monte Santo, the history of Aicuña occurring in 1674, 1723, 1833, 1866, in the late 1890s (Manzoli et al., 2013). 1912, 1955, and 1971 for which good written documentation exists. The information provided in these documents Aicuña (example 3): The sudden flood in 1952, de- allowed the construction of the basic genealogy of Aicuña scribed in detail in the historical book of Aicuña, merits since 1610, the approximate date of birth of the recognized comment because it was the largest catastrophe described founder of the kinship, General Pedro Nicolás de Brizuela. in the history of Aicuña. It occurred around sunset on the With the exception of the interval between the judgment of 19th of January and was caused by torrential rain in the 1723 and the successor agreement of 1833, the intervals hills surrounding Aicuña. The torrents of water and large between all other dates are less than 50 years, or one or two rocks destroyed plantations, irrigation works and even generations. Consequently, much of the information ob- houses, although no one was injured or killed. The signifi- tained from consecutive events overlaps. This not only fa- cance of the record of this catastrophe is that it serves as a cilitated the construction of the genealogy but also ensured point of reference from which we deduce that nothing more its accuracy. The interval of 110 years between 1723 and serious occurred in Aicuña during 350 years of 1833 includes four generations, but at the time the popula- well-documented history. Thus, the history of this isolate tion size was very small. differs in this respect, for example, from that of Tristan da Fiction Cunha (Thompson, 1978). From a genetic structure stand- Some families and populations are described or incor- point, catastrophes are relevant as long as they substan- porated into fictional literature, either directly or indirectly. tially reduce the effective population size, creating Among the former, the population of Ricaurte, in the Valle conditions for genetic drift to occur and affecting one or del Cauca Department, Colombia, a cluster for fragile X more mutated genes. (Wilmar Saldarriaga, personal communication), was the or-

Table 2 - Historical facts in the Aicuña population ensuring a recorded population pedigree extended through 15 generations, along 297 years.

Year Fact 1674 The land of Aicuna was purchased by the founder of this kinship, who immigrated from Spain in 1630, and given as a deed of gift to his illegitimate son. The scripture is filed in the court house of La Rioja. It covers the first two generations: birth years 1610 and 1650. GAP (Years: 49 / Generations: 2) 1723 A granddaughter of the founder and her husband won a trial against influential people trying to take the land away from the legal heirs, whose rights are based on pedigree data. The trial record is filed in the court house of La Rioja. It includes a third generation, with its birth-year about 1680. GAP (Years: 110 / Generations: 4) 1833 The land was divided in two parts, Aicuña and Tambillos, through a successory agreement signed by members of the seventh generation of the kinship. This issue was based on well recorded detailed genealogical data. The agreement record is filed in the court house of La Rioja. It extends the pedigree up to a 7th. generation, with a birth-year about 1780. GAP (Years: 33 / Generations: 2) 1866 For an unknown reason, a local priest named Fray Aymon wrote a detailed pedigree expanding the one made in 1833 a couple of generations further. This document is kept in the parish of Villa Union. The pedigree is extended to a 9th generation, with a birth-year about 1840. GAP (Years: 46 / Generations: 2) 1912 A new successory agreement was signed among the heirs of the land of Aicuna, including further genealogical data. The agreement record is filed in the court house of Chilecito. The pedigree reaches now to an 11th generation, with a birth-year about 1890. GAP (Years: 43 / Generations: 2) 1955 A commission was created to update the pedigree, to list out the declared heirs and to carry on a new successory agreement, which was finally performed in 1963. Pedigrees and declaration of heirs is kept by commission members of Aicuña. Pedigree extended to a 13th generation, with a birth-year around 1920. GAP (Years: 16 / Generations: 1) 1971 The authors expanded the pedigree up to births occurred in 1971, which do approximately correspond to members of the 15th generation of the kinship. Both, written and oral sources of data were used. Genealogical data 179 igin of El Divino (Alvarez-Gardeazábal, 1986), a novel usually not linked to any cultural anathema and so does not also produced for TV. Seemingly fictional genealogies, need to be hidden in Latin America at least (Jeambrun and such as the Terras-Cambarás in “O Tempo e o Vento” by Sergent, 1991). The alternative situations presented by dis- Erico Verissimo (1984), and the Buendias in Cien Años de eases as galactosemia, ataxia-telangiectasia, Huntington’s Soledad by Gabriel García Márquez (1973) most likely rep- chorea, etc. are easy to identify. resent the reality of whole populations (Castilla and Ad- BY PLACE OF RESIDENCE OR DEATH: inclusion ams, 1996). Even outside of Latin America, large pedigrees or elimination of emigrated family branches. served as inspiration for well-known novels as The Tin Search for sources of information Drum by Günter Grass (1999). List available informers, oral and written, their local- Pedigree construction ization, validation of reliability, and expected usefulness of Depending upon the programmed plan, one or more each source for specific sections and procedures in con- operators might be simultaneously be collecting genealogi- structing the genealogy. cal data from one or more informers. In a simple example, Steps: Initial, extension, repairs while one person is working at the parish house searching Define specific steps to be taken, assigned operators, for specific information concerning consanguinity type and estimate timing, decide operative places, select dates, etc. degree of specific marriages, a second colleague may be collecting the pedigree of a family group from two oral in- Initial formers. Decide on your strategy for index cases. They could Field methods be persons affected by a certain disease, the youngest member, or the eldest member of that family group, etc. Feel free These are tasks to be organized for performance at the to decide according to your preset objectives (see Pedigree population site, that is, in the field. objectives ), and general planning, but make a decision and Preliminary consultations stick to it. Search for suitable informers, including available Extension documentation, and suitable verbal informers. Decide on the direction (see Section Pedigree expan- Prioritize well-informed elders because they may not sion, below) and extent of pedigree progression. For in- be available at your next site visit. stance, you can work with an oral informer to generate Working definitions for space, time, and persons pedigree data by ascent until the eldest ancestors this informer knows, leaving these data to be linked with informa- Space, time, and persons are the classical three pillars tion on elder generations obtained or to be obtained from of epidemiology (Porta, 2008). written documents. The linkage, if successful, is defined as SPACE. Define areas for demographic purposes of a junction or intersection (entronque). the population, as well as for related definition such as migration patterns. Covering progressively concentric areas Repairs can be a useful practical approach. Review the collected information and search for TIME. Do not register ages but rather year of birth be- omissions and inconsistencies to be repaired in the next en- cause age is relative to the time of data recording. Families counter with the same informers. Some omissions may re- and populations as breeding units are nearly eternal. Thus, quire the interview of other or more informers. The affected define the intended time extension ahead of time, for in- information can be genealogical (parentage; gametic links) th stance, the 18 century if the founder generation is esti- or personal, the former being more critical because it does mated to have entered the region around the year 1700. not affect only a single individual. Compare data from dif- PERSONS. Decide the type of individuals or pedi- ferent informers (See Section Pedigree assembling, below). gree members to be included. BY SURVIVAL: induced abortions, spontaneous miscarriages, stillbirths, neonatal Pedigree assembling deaths, etc. BY DISEASE: affected, unaffected, or un- Perhaps the most time consuming component of the known. Special attention must be given to the unknown, assembly of the data set involving the linking of multiple which can be easily confused with unaffected when the dis- records is putting together information gathered by differ- ease is not conspicuous or congenital, is highly lethal, or is ent operators from different informers. This work involves a local taboo. In any of these situations, the estimated three aspects, resolution of different sources of informa- penetrance of a mutant gene can be underestimated or even tion, linkage of genealogical information with demo- undetected. OCA (Oculo-cutaneous albinism) is a good ex- graphic information, and linkage of records of nuclear ample of an easily recognizable phenotype because it is ob- families. vious to any person without requiring a special examina- The existence of multiple sources of information tion; it is present at birth, it survives to adulthood, and it is makes it possible to construct an unusually reliable and 180 Poletta et al. complete data set. However, this inevitably means that for Aicuña (example 4): The linkage of nuclear families some individuals, discrepancies and inconsistencies have in the genealogy was performed manually. The basic gene- to be solved. While many discrepancies disappear after alogy elaborated during the 18th and 19th centuries was careful review of the records and are revealed as simple er- used as the basic frame of reference because it contained rors in transcription, others are not so easily worked out. In the largest numbers of individuals. However, “biological” such cases, further sources of information should be sought logic required that links were established descending the out and investigated. If the discrepancy remains, which oc- pedigree in contrast to the usual approach of ascending the curs in only a small minority of cases, the data must be dis- pedigree from the contemporary population toward the an- carded and/or recorded simply as missing or unknown. cestral founders. Initially, a branch of the genealogy was To validate means to ratify or rectify the genealogical ascertained by descent and then verified by ascent, prefera- information regarding the identity of a person, his/her bly using independent sources of information. The over- mother and father; whenever possible, at least three inde- whelming predominance of a few last names and a pendent sources of information for each gametic link tendency to repeat first names in a certain branch or the (mother-child and father-child) are to be collected. popularity of certain first names in a given generation oc- casionally made the task of record linking quite difficult. In Pedigree expansion such cases, the use of nicknames as identifiers proved to be Ascent invaluable. These nicknames were often derived from some unusual physical characteristic, such as La Rubia (the This phase of the survey follows the family in a direct Blonde), El Turco (literally the Turk, but also an epithet line through an ancestor to the oldest known genealogy commonly used in the area to refer to someone with a for- members. Ancestors are followed upwards in a direct line, eign appearance), a particular handicap, such as El Rengo without sidewise extensions. (the limper), some official capacity the individual may This procedure intends to disclose possible genealog- have, such as El Aguatero (the Waterer), or El Caminero ical links among not obviously related family groups. Fur- (the road worker). thermore, it simultaneously searches for genealogy founders. Biological validation of pedigree data One of the effects of inbreeding is reduced ancestors. Because pedigree data are based on feeble sources, “Reducing families” is another way of seeing the same re- the resulting genealogies should be validated with biologi- duction. The more families are linked in their origin, the cal facts, whenever possible. In the case of the Aicuña pop- easier it will be to define the breeding structure for that population, an 85 % agreement was found between the biologi- ulation or family (See Section Four basic sub-routines, be- cal and genealogical data (Bailliet et al., 2001). low). Databases with genealogical data Descent Database construction This procedure is used when one or more crucial breeders are identified. A crucial breeder can be a person As described in the previous sections, genealogical identified for a given pedigree as the founder, a migrant to data could be collected by the tabulated method using paper another location from whom a family branch and a new lo- recording forms. These genealogical data should then be cality is derived, a survivor of a catastrophe seeding many keyed into databases for electronic storage, quality control, offspring, etc. and analysis. A simple spreadsheet application such as Calc, Excel, Lotus 1-2-3, etc. or a database management Sideways program such as Base, MySQL, Fox-Pro, Access, or simi- Sideways genealogical data collection, that is, ex- lar, could be used to design these databases. tending it to siblings of the ancestors and their descendants, When a single pedigree is considered, each record is mainly used in order to identify related persons with a represent one pedigree member that is identified by an indi- given phenotype, such as a disease for instance. Otherwise, vidual ID, while the triad IDs (individual, father, and those data can be summarized, leaving them for later data mother) and the connections among nuclear families define completion or not. the degree of relationship among all individuals in the pedigree (See Table 1 and Figure 1). Linkage of nuclear families within the genealogy of the However, these individual IDs may not be adequate population as unique identifiers when several families are recruited Linkage among nuclear families is usually performed into the study and are recorded in the genealogical data- manually, although when a very large pedigree is to be han- base. Thus, another unique identifier, called the “key vari- dled, the process can be programmed and executed elec- able” is then necessary to identify each individual within tronically. Nevertheless, manual terminating is recom- the overall databases. This ”key" identifier could be, for ex- mended. ample, a combination of the pedigree ID and the Individual Genealogical data 181

ID (PID+IID), or a unique alphanumeric identifier that is name, a Directory. Having this printed output on hand not related to the pedigree IDs. The latter would be recom- makes the reconstruction of a given person’s genealogy mended to maintain a higher level of con?dentiality with re- simple and fast even in the absence of an electronic device. spect to certain labeled materials and within databases that The names of the required person are sought in the Direc- include names and disease and genotype information. tory, the ID number notated, and then the full record is In addition, this key variable is useful to interconnect searched in the Pedigree, where the parental IDs are also data from several databases associated with a research pro- written. Pedigree ascent can then be easily performed fol- ject including personal information, namely, demograph- lowing successive parental numbers. If descent is desired, ics, medical examinations, phenotypes, family medical the original person’s ID number is pursued in the corre- history, perinatal history, genotypes, etc. Information con- sponding column, Father or Mother, according to the gen- cerning each individual stored as single records in different der. This procedure can be more troublesome when databases could then be linked using this key variable, working manually if individuals have not been identified bringing different data sets together into customized job with consecutively numbered IDs and the parent columns files according to specific requirements for queries and data are not ordered numerically. With the help of known family analysis (Figure 2). groups, last names, or localities, this search is possible, al- Four basic sub-routines though not easy. Nevertheless, this procedure is facilitated with the use of available computational resources, some of Four main outputs indicating different aspects of the which are described below in Section Software for pedigree pedigree breeding structure can be produced, as indicated analysis (below). by McLean (1969): - By ascent: tracing all direct ascendants from any Population genetic structure given person in the pedigree, designed as ancestry, kinship, origin, parentage, extraction, or root. Two different population units that partially overlap - By descent: providing all direct descendants from should be considered, namely, the Genealogical and the any given person in the pedigree: clone, heritage, legacy, or Demographic. The first one, also designated as the “histori- lineage. cal” population, can be defined as all the descendants of a - Inbreeding: calculating the inbreeding coefficient given founding person, couple, or group of persons, dead or for any person in the pedigree, including parental consan- alive, emigrated or not, while the second is defined as all guinity. persons living within a defined geographic area at a given - Relationship: computing the coefficient of relation- time, including but not exclusive of the descendants of a ship between any pair of persons in the pedigree, also given founding person, couple, or group of persons. For in- known as parentage or family links. stance, in the case of Aicuña, the genealogical population Pedigree outputs included over 8,000 persons, while the demographic population at the time of the study comprised approximately 400 Pedigree and directory inhabitants for the strict area, the village, and 2,000 for the Dataset records ordered numerically by ID numbers broader territory, the “estancia” (Castilla and Adams, are a Pedigree, and when they are ordered alphabetically by 1990).

Figure 2 - A “key” variable enables the merging of different data sets into customized job files according to specific requirements for data analysis. 182 Poletta et al.

Pedigree analysis multifactorial threshold model of inheritance (Reich et al., The approaches chosen for the analysis of genealogi- 1972). cal data will depend on the questions being asked in each Pedigree data can also be used to measure phenotypic aim of the research study. Thus, with appropriate design correlations by degree of relationship (pairs of relatives and analysis, it would be possible estimate the population with concordant and discordant phenotypes). This allows breeding structure; evaluate the existence of genetic factors us to estimate the proportion of the total phenotypic vari- associated with disease susceptibility in these families; and ance that is due to inheritance of genetic factors in combination with genotyping data, detect causative (heritability) (Emery, 1986) and evaluate whether this pat- genes through linkage and family-based association analy- tern of correlation is consistent with a possible genetic ef- sis. fect. In a descriptive analysis of the reproductive structure of the population, we could trace and list all direct ascen- If positive evidence of familial aggregation and a pat- dants or descendants from any given person in the pedi- tern of correlation consistent with a possible effect of genes gree, select a subset of individuals who have a common an- has been found, the next step is to search for the effect of a cestor, or find individuals with multiple mates or major gene segregating in these families. This kind of sta- consanguineous mating pairs. Moreover, it would be in- tistical analysis can also be performed using pedigree data, teresting to estimate some indicators such as the inbreeding which is known as complex segregation analysis (CSA) coefficient (f or F). It could be defined as the coefficient of (Lalouel et al., 1983). Briefly, CSA is a more elaborate correlation between alleles of uniting gametes for a individ- method to compare the statistical fit of the observed pedi- ual (Wright, 1922), relative to gametes drawn at random gree data with alternative and more complex models of in- from a population; or as the probability that two homolo- heritance, such as sporadic, environmental, multifacto- gous alleles in an individual are identical by descent (IBD) rial/polygenic, Mendelian major gene, and several mixed (Malécot, 1948), that is to say, they are derived from one al- models. Different parameters that define each alternative lele of a common ancestor. This could be estimated from model are estimated through the maximum-likelihood pedigree data using Wright’s path method (Wright, 1934). method: the frequency of the high-risk allele (q) at the ma- Additionally, the coefficient of relationship (r) (Wright, jor gene (inferred from phenotype segregation in pedi- 1922), also known as kinship coefficient or relatedness be- grees), the allele transmission probabilities (1.0; 0.5; and 0 tween two individuals, is the probability that genes ran- for the three genotypes in Mendelian models), and the domly selected from an individual “A” and from an indi- multifactorial heritability (the additive influence of poly- vidual “B” are IBD (Falconer and Mackay, 1996), so it is genic background different from the major gene effect). equal to twice the inbreeding coefficient of their possible These parameters can then be utilized in parametric linkage offspring (r =2f). These indicators are frequently used as a analysis or to used estimate the power and sample size in measure of the level of homozygosity and are useful predic- family-based association studies. In CSA, there are specific tors of covariance and phenotypic correlation between rela- analytical approaches based on the characteristics of the tives. They can be expressed per individual, as the popula- trait under study (for example, a qualitative or quantitative tion average, or as the average for some particular groups trait); two of the most employed approaches for the study of such as affected and unaffected, founders and non- diseases are the unified mixed model (Lalouel and Morton, founders, or by generations. 1981) and the regressive multivariate logistic model for bi- When the disease under study has a complex etiology, nary traits (Karunaratne and Elston, 1998). one of the first steps in the approach is to determine whether Starting from databases, as defined in Section Data- there is evidence of familial aggregation and a possible ef- bases for genealogical analysis (above), genealogical data fect of genes. Pedigree data can be used to calculate the fre- will be set up for these types of queries and statistical analy- quency of the disease by the degree of relationship with the sis. As outlined below, several computational resources to probands and estimate recurrence patterns (Davie, 1979; carry out pedigree analyses are available. Khoury et al., 1993) and familial relative risks (Risch, 1990) before genotyping is done. Familial relative risks Software for pedigree data handling and analysis (FRRs) or relative recurrence risk (RRR) for a relative of type R (lR) are calculated as the ratio of the risk of recur- We briefly describe here some software and computa- rence to a relative, R, and the risk in the population (base- tional packages for data handling and pedigree analysis. In line prevalence). The patterns of familial recurrences and each case, the formats of input and output files are shown. the drop off in the observed FRR from the first (l1), to sec- As an example, data files from the Aicuña research ond (l2), and third-degree (l3) relatives can potentially project will be used. A subpedigree with 196 individuals suggest different models of inheritance based on the ex- has been extracted from the complete genealogy of Aicuña pected values for a single major locus, different multiplica- (8,696 individuals) and is available for download as supple- tive multilocus (epistatic) models (Risch, 1990), and for the mentary material in an ASCII text file (File S1). Genealogical data 183

Pedigree drawing classical four outputs “Ascent”, “Descent”, “Inbreeding”, Several pedigree-drawing software programs are and “Relationship” originally implemented in a computer available for use in medical population genetics, including program by McLean (1969) can be now calculated from the following: COPE (COllaborative Pedigree drawing En- several available and user-friendly software programs. vironment) (Brun-Samarcq et al., 1999), Cyrillic (Chap- Table 3 shows a detailed description of pedigree sta- man, 1990), functions run in R such as plot.pedigree in the tistics and breeding structure of the genealogy with 196 in- package kinship (Sinnwell and Atkinson, 2013), dividuals in File S1 and plotted in Figure S1. We will pedigreemm, pedigree, and pedtodot in the package gap briefly describe here some available software programs that (Zhao, 2007), Haplopainter (Thiele and Nürnberg, 2005), can be used to calculate these and other indicators. Madeline (Trager et al., 2007), Pedigree/Draw (Mamelka A general description of the pedigree can be obtained et al., 1987), PedigreeQuery (Kirichenko, 2004), by running the program PEDINFO available in S.A.G.E. PedHunter (Agarwala et al., 1998), and Progeny (Progeny version 6.2 (2012). To import the data to S.A.G.E., the in- Software LLC, South Bend, Indiana, USA), among several put file must have the format shown in File S3. Programs in others. S.A.G.E. may be executed by means of a command line di- As an example, the process for plotting the genealogy rective or from the provided Graphical User Interface in File S1 using Progeny is as follows: (1) Download the (GUI) as follows: tab-delimited text File S1; (2) Unfold the menu options and Table 3 - Analysis of pedigree structure and inbreeding of a subpedigree select: Pedigrees > Import... >; (3) In the Import module, with 196 individuals from the Aicuña research project. browse the file and then select the File Delimiter (“tab”); 1 the Import Type (“Pedigrees”), and enter the values that Affected Unaffected Total used in the database for Male, Female, and Deceased; and Pedigrees - - 1 select the columns names. The pedigree will automatically Individuals 19 145 196 be drawn, adding missing parents. For complex pedigrees Gender: Male 9 71 94 with loops, multiple matings and consanguineous matings, Female 10 74 102 manual edition of the pedigree could be necessary. The Founder 0 29 61 graphical pedigree corresponding to the tabulated genealogy in File S1 is shown in Figure S1. Nonfounder 19 116 135 An alternative to commercial software is to use func- Consanguineous mating pairs - - 11 tions for pedigree drawing under the GNU General Inbreeds 9 17 26 PublicLicense, such as packages available in R. For exam- Average inbreeding coeff. 0.016 0.034 0.028 ple, the package called kinship (and kinship2) can be used Average inbreeding coeff. (in 0.0075 0.0039 0.0037 to plot the tabulated genealogy in File S1. The format of the the inbreds) input file should be as shown in File S2. Call the package by Max inbreeding coeff. 0.0376 0.0625 0.0625 typing the following at the shell prompt: Min inbreeding coeff. 0.0039 0.0078 0.0039 >R Inbreeding coeff. (f) distribu- > install.packages(“kinship2") tion: > library(kinship2) 0.00 aicped < - read.table(“C:/your_current_direc- tory/file_s2.txt”, header=T, sep="\t") 0.05 attach(aicped) 0.10 ped < - pedigree(id, fid, mid, sex, aff) > par(xpd=T) Longest ancestral path (LAP): 0 - - 60 > plot.pedigree(ped, cex=0.45, symbolsize=1.5) 1to5 - - 27 This function gives a graphic object (Plots) that can 6to10 - - 104 be saved as image format and is shown in Figure S2. A de- 11 to 12 - - 5 tailed description of the arguments and options available in Pairs2: Concordant Concordant this package can be obtained typing ”?plot.pedigree” or Aff. Unaff. “?kinship2" at the shell prompt. Parent/Off 0 201 270 More information about other software for drawing Sib/Sib 14 204 218 pedigrees is available in “An alphabetic list of Genetic Grandparents 0 228 318 Analysis Software” (http://www.jurgott.org/link- Avuncular 0 438 438 age/ListSoftware.html). Half Sib 0 12 12 Pedigree description Cousin 0 912 912

As detailed in Section Pedigree analysis (above), sev- (1) Includes missing/unknown data; (2) Pairs of relatives that show con- eral statistics can be calculated to describe a pedigree. The cordant or discordant phenotype (affected or unaffected). 184 Poletta et al.

First, in the “Setup Dialog” window, select “Create Ethics for field work new project”, and choose a project name. In the next step, As discussed before (Section Illegitimacy, confiden- select the option “I have all data required by S.A.G.E. pedi- tiality and other ethical issues), the right of a proband to gree but not parameter file”. Browse the directory to where confidentiality conflicts with the carrier’s right to be in- the input file is located in your computer, select the format formed when seeking prospective counseling. Discussions file (“tab” and “single” in the File S3), and import the data and recommendations on these issues can be found in the file. The data file is now available as S.A.G.E. internal data WHO report of a consultation on community genetics in (see folder “Data” on the left) and can be used to run low- and middle-income countries, section 7.2 Confidenti- PEDINFO and other S.A.G.E. programs such as SEGREG ality issues, (WHO, 2010, pp 16). for complex segregation analysis or the LODLINK and MLOD programs for model-based linkage analysis. Concerning the use of written genealogical sources To run PEDINFO, in the menu window, select Analy- such as churches, civil, electorate, military, etc., Brazilian sis > Summary Statistics > PEDINFO, and then complete legislation (Resolução CNS 466/2012) does not require the directory path of data file and the name of output file. In IRB approval for data obtained from public databases. “Analysis Definition”, select whether you want statistics for a particular trait or covariate and/or statistics for each Acknowledgments pedigree. Run it. An output folder will be created in the This project was supported by grants from INCT- left-hand section of the windows that includes the output INAGEMP; Ministry of Science and Technology/CNPq, files with the results of the analysis. Supplementary mate- Brazil, grant no. 573993/2008; CNPq, Brazil, grants # rial File S4 shows the PEDINFO results for the same pedi- 402045/2010-6, 481069/2012-7, 306396/2013-0; CNPq, gree analyzed as an example. Programa Ciência sem Fronteiras (CSF), modalidade AJT, Another user-friendly program used in population ge- Brazil, processo # 370799/2013-5; FAPERJ, Brazil grant # netics for pedigree analysis is CFC (Colleau, 2002), which E-26/102.797/2012; CONICET, National Research Coun- can provide general information on the structure of pedi- cil of Argentina; ANPCyT, Argentina, grant # PICT 2010- grees, check for pedigree errors, extract a list of ancestors 2798. No funding bodies had any role in the study design, and descendants, draw the paths that connect relatives of in- data collection and analysis, decision to publish, or prepa- bred individuals, and compute indicators such as inbreed- ration of the manuscript. ing coefficients, coefficient of relationships, ancestral decomposition of inbreeding coefficients, probabilities of References gene origin, etc. When starting to use CFC, a pedigree input file Agarwala R, Biesecker LG, Hopkins KA, Francomano CA and should be formatted as shown in Supplementary Material Schaffer AA (1998) Software for constructing and verifying File S5. To open the pedigree file, select File > Open, and pedigrees within large genealogies and an application to the select the input file. The pedigree file will be read, checked Old Order Amish of Lancaster County. Genome Res 8:211- 221. for errors, and saved in “pgd” extension. Alvarez-Gardeazábal G (1986) El Divino, Plaza & Janés. Bogotá, It is possible to obtain a brief structure of the pedi- 233 pp. gree, including the distribution according to the inbreeding Bailliet G, Castilla EE, Adams JP, Orioli I, Martínez-Marignac V, coefficients, the longest ancestral path (LAP), number of Richard SM and Bianchi N (2001) Correlation between mo- founders, number of inbreeds, average of inbreeding coeffi- lecular and conventional genealogies in Aicuña: A rural cient, etc., by selecting Tools > Pedigree Structure. population from Northwestern Argentina. Hum Hered The results of these analyses can be exported as a .txt 51:150-159. file, and the output file for the example analyzed is shown Bennett RL, French KS, Resta RG and Doyle DL (2008) Stan- in Supplementary Material File S6. All other analyses can dardized human pedigree nomenclature: Update and assess- be run from the “Tools” menu. ment of the recommendations of the National Society of Ge- Others sophisticated statistical packages for pedigree netic Counselors. J Genet Counsel 17:424-433. analysis are available, including Genetic Analysis Package Bronberg RA, Dipierri JE, Alfaro EL, Barrai I, Rodriguez-Lar- (GAP) (Zhao, 2007), PedHunter (Agarwala et al., 1998), or ralde A, Castilla EE, Colonna V, Rodriguez-Arroyo G and Bailliet G (2009) Isonymy structure of Buenos Aires city. Madeline (Trager et al., 2007), but the description of the Hum Biol 81:447-461. mandatory formats for input files and the arguments to exe- Brun-Samarcq L, Gallina S, Philippi A, Demenais F, Vaysseix G cute the specific commands are beyond the aims of this and Barillot E (1999) CoPE: A collaborative pedigree draw- manuscript. ing environment. Bioinformatics 15:345-346. We hope that this fieldwork guide for collecting, han- Castilla EE and Adams J (1990) Migration and genetic structure in dling and analyzing genealogical data will help to design an isolated population in Argentina: Aicuña. In: Adams JP research projects in population medical genetics and pro- (ed) Proceeding of Convergent Issues in Genetics and De- mote the study of isolated populations in Latin America. mography. Oxford University Press, New York, pp 45-62. Genealogical data 185

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Vargas DM, Arena LFGL and Soncini AS (2010) Secular trends Greven PJ (1972) Four Generations: Population, Land and Family in stature growth in Blumenau-Brazil in relation to human in Colonial Andover, Massachusetts. Cornell University development index (HDI). Rev Assoc Med Bras 56:304- Press, Ithaca, 349 pp. 308. Jeambrun P and Sergent B (1991) Les Enfants de la Lune: L’albi- Veríssimo É (1984) O Tempo e O Vento. Editora Globo, Porto nisme chez les Amérindiens. IRD Editions, Montpellier, Alegre. 351 pp. WHO (2010) World Health Organization: Community genetics Karunaratne PM and Elston RC (1998) A multivariate logistic services: Report of a WHO consultation on community ge- model (MLM) for analyzing binary family data. Am J Med netics in low-and middle-income countries. World Health Genet 76:428-437. Organization, Geneva. Wright S (1922) Coefficients of inbreeding and relationship. Am Khoury MJ, Botto L, Waters GD, Mastroiacovo P, Castilla E and Nat 56:330-338. Erickson JD (1993) Monitoring for new multiple congenital Wright S (1934) On the method of path coef?cients. Ann Math anomalies in the search for human teratogens. Am J Med Stat 5:161-215. Genet 46:460-466. Zhao JH (2007) gap: Genetic analysis package. J Stat Softw Kirichenko A (2004) An algorithm of step-by-step pedigree draw- 23:1-18. ing. Russ J Genet 40:1176-1178. Lalouel JM and Morton NE (1981) Complex segregation analysis Internet Resources with pointers. Hum Hered 31:312-321. Lalouel JM, Rao DC, Morton NE and Elston RC (1983) A unified S.A.G.E. (2012) Statistical Analysis for Genetic Epidemiology. model for complex segregation analysis. Am J Hum Genet Release 6.3. http://darwin.cwru.edu/. 35:816-826. MacLean C (1969) Computer analysis of pedigree data. In: Mor- Supplementary material ton NE (ed) Computer Applications in Genetics. University The following online material is available for this article: of Hawaii Press, Honolulu, pp 82-86. - Figure S1 - Graphical pedigree with 196 individuals correspond- Malécot G (1948) Les Mathématiques de l’Hérédité. Masson, ing to the tabulated genealogy in File S1. Paris, 63 pp. - Figure S2 - Graphical pedigree that is the output file of kinship2 Mamelka P, Dyke B and MacCluerJ (1987) Pedigree/Draw for the (R package). Apple Macintosh. Department of Genetics, Southwest - File S1 - Tabulated genealogy of subpedigree with 196 individu- Foundation for Biomedical Research, San Antonio. als extracted from the complete genealogy of Aicuña (8,696 individuals). Manzoli GN, Abe-Sandes K, Bittles AH, Da Silva DS, Fernandes - File S2 - Input file format for drawing pedigrees using kinship2 LDC, Paulon R, De Castro ICS, Padovani CM and Acosta (R package). AX (2013) Non-syndromic hearing impairment in a multi- - File S3 - Input file format for pedigree analysis using S.A.G.E. ethnic population of Northeastern Brazil. Int J Pediatr - File S4 - Output file of PEDINFO program included in S.A.G.E. Otorhinolaryngol 77:1011-1082. - File S5 - Input file format for pedigree analysis using CFC. McCaa R (1983) Marriage and fertility in Chile: Demographic - File S6 - Output file with the “Pedigree structure” using CFC turning points in the Petorca Valley, 1840-1976. Westview software. Press, Boulder, 207 pp. th License information: This is an open-access article distributed under the terms of the Porta M (2008) A Dictionary of Epidemiology. 5 edition. Oxford Creative Commons Attribution License, which permits unrestricted use, distribution, and University Press, New York, 316 pp. reproduction in any medium, provided the original work is properly cited. MS 16 - suppl mat - file_s6.txt 10:44 Thursday, January 11, 2014 Pedigree file name: aic_gemepo_v3_CFC.txt Brief structure of the pedigree: Number Individuals in total 196 Inbreds in total 26 Evaluated individuals 196 Inbreds in evaluated 26 Sires in total 65 -Progeny 136 Dams in total 66 -Progeny 135 Individuals with progeny 131 Individuals with no progeny 65 Founders 60 -Progeny 109 -Sires 24 -Progeny 49 -Dams 34 -Progeny 63 -With no progeny 2

Non-founders 136 -Sires 41 -Progeny 87 -Dams 32 -Progeny 72 -Only with known sire 1 -Only with known dam 0 -With known sire and dam 135

Full-sib groups 24 -Average family size 3.83333 -Maximum 12 -Minimum 2

Distribution of inbreeding coefficients ------0.00 < F <= 0.05 23 0.05 < F <= 0.10 3 0.10 < F <= 0.15 0 0.15 < F <= 0.20 0 0.20 < F <= 0.25 0 0.25 < F <= 0.30 0 0.30 < F <= 0.35 0 0.35 < F <= 0.40 0 0.40 < F <= 0.45 0 0.45 < F <= 0.50 0 0.50 < F <= 0.55 0 0.55 < F <= 0.60 0 0.60 < F <= 0.65 0 0.65 < F <= 0.70 0 0.70 < F <= 0.75 0 0.75 < F <= 0.80 0 0.80 < F <= 0.85 0 0.85 < F <= 0.90 0 0.90 < F <= 0.95 0 0.95 < F <= 1.00 0 ------

Page 1 MS 16 - suppl mat - file_s6.txt Longest ancestral path (LAP): 0 60 1 4 2 2 3 5 4 4 5 12 6 43 7 21 8 13 9 12 10 15 11 3 12 2 A: Number of individuals B: Number of inbreds C: Number of founders D: Number of individuals with both known parents E: Number of individuals with no progeny

Group A B C D E ------Total 196 26 60 135 65 1 145 17 29 116 43 2 19 9 0 19 19 ------

F: Number of unique ancestors for the group (excluding ancestors in the group) G: Average inbreeding coefficients H: Average inbreeding coefficients in the inbreds I: Maximum of inbreeding coefficients J: Minimum of inbreeding coefficients

Group F G H I J ------Total 0 0.00367457 0.0277006 0.0625 0.00390625 1 112 0.00398707 0.0340074 0.0625 0.0078125 2 122 0.00747841 0.0157878 0.0375977 0.00390625 ------

K: Average numerator relationships (Reciprocals + Self-relationships) L: Average numerator relationships (Self-relationships are excluded) M: Average number of discrete generation equivalents (or average of average number of generations) N: Maximum number of discrete generation equivalents O: Minimum number of discrete generation equivalents Group K L M N O ------Total 0.0478011 0.0428992 1.51483 3.85352 0 1 0.0718707 0.0653976 1.67882 3.46875 0 2 0.100179 0.0497732 2.78829 3.85352 1.99805 ------

Page 2 MS 16 - suppl mat - file_s5.txt Progeny Sire Dam Group F 41 63 2295 1 . 55001. 56001. 57001. 58001. 59 9244 74 1 . 60001. 61 73 75 1 . 62 73 75 1 . 63 73 75 1 . 64 73 75 1 . 65 73 75 1 . 66 73 75 1 . 67 73 75 1 . 68 73 75 1 . 69 76 1672 1 . 70 76 1672 1 . 71 69 1673 1 . 72 69 1673 1 . 73001. 74 77 79 1 . 75 77 79 1 . 76 81 1671 1 . 77001. 78 82 83 1 . 79 82 83 1 . 80 82 83 1 . 81 82 83 1 . 82001. 83 7681 9245 1 . 553 62 58 1 . 554 62 58 1 . 555 62 58 1 . 556 62 58 1 . 557 62 58 1 . 558 62 58 1 . 559 62 58 1 . 801 67 1683 1 . 802 67 1683 1 . 803 67 1683 1 . 804 67 1683 1 . 805 67 1683 1 . 806 67 1683 1 . 819 56 64 1 . 820 56 64 1 . 821 56 64 1 . 822 56 64 1 . 823 56 64 1 . 826 56 64 1 . 1017 60 66 1 . 1018 60 66 1 . 1019 60 66 1 . 1183 001. 1189 1183 61 1 . 1671 001. 1672 001. 1673 001. 1683 001. 2269 9999 61 1 . 2279 55 65 1 . 2280 55 65 1 . 2281 55 65 1 . Page 1 MS 16 - suppl mat - file_s5.txt 2290 59 68 1 . 2291 59 68 1 . 2292 59 68 1 . 2293 59 57 1 . 2294 59 57 1 . 2295 59 57 1 . 2306 63 2295 1 . 2307 63 2295 1 . 2308 63 2295 1 . 2309 63 2295 1 . 2310 63 2295 1 . 2311 63 2295 1 . 2312 63 2295 1 . 2313 63 2295 1 . 2314 63 2295 1 . 3375 67 1683 1 . 3382 67 1683 1 . 3383 67 1683 1 . 3384 67 1683 1 . 3385 67 1683 1 . 3386 67 1683 1 . 7681 7682 7683 1 . 7682 001. 7683 001. 7684 001. 7685 7682 7684 1 . 7686 001. 7687 7685 7686 1 . 7688 001. 7689 7688 7687 1 . 7690 001. 7691 7689 7690 1 . 7692 001. 7694 7691 7692 1 . 7695 001. 7698 7695 7696 1 . 7699 001. 7700 7698 7694 1 . 9999 001. 7696 001. 9109 00.. 9134 9135 0 . . 9135 00.. 9138 00.. 167 71 161 1 . 183 167 9246 1 . 185 183 9247 1 . 525 185 9248 2 . 527 185 9248 2 . 529 185 9248 2 . 161001. 1227 1648 70 1 . 1667 1227 9249 1 . 1225 1227 9249 1 . 1224 1227 9249 1 . 2321 1667 9250 1 . 1111 2321 9251 1 . 1714 1111 9252 1 . 1842 1714 1835 1 . 2920 1842 2908 2 . 2921 1842 2908 2 . 35 9256 1224 1 . 1231 9256 1224 1 . Page 2 MS 16 - suppl mat - file_s5.txt 1235 9256 1224 1 . 2886 35 9257 1 . 2908 9258 2886 1 . 1835 1235 566 1 . 566001. 2325 1231 2320 1 . 2 2325 9259 2 . 1419 2325 9259 2 . 2320 001. 2511 001. 2619 1231 2511 1 . 2622 9260 2619 1 . 1901 9254 1225 1 . 2261 1901 9255 1 . 2646 2261 2622 2 . 2648 2261 2622 2 . 1648 001. 899 9263 822 1 . 901 9264 899 1 . 1399 901 1211 2 . 1394 901 1211 2 . 1191 1189 9262 1 . 1211 1191 9261 1 . 1005 9266 826 1 . 1007 1005 9265 1 . 800 1007 771 2 . 763 9267 2310 1 . 771 763 1027 1 . 1027 1017 9268 1 . 1073 9269 1018 1 . 1077 2307 1073 1 . 2317 9270 2309 1 . 2443 2317 9271 1 . 2446 1077 2443 2 . 2447 1077 2443 2 . 1071 9269 1018 1 . 2506 9267 2310 1 . 2507 1071 2506 1 . 1451 2507 9272 2 . 1454 2507 9272 2 . 1458 2507 9272 2 . 1459 2507 9272 2 . 7701 7700 7699 2 . 9244 00.. 9245 00.. 9246 00.. 9247 00.. 9248 00.. 9249 00.. 9250 00.. 9251 00.. 9252 00.. 9254 00.. 9255 00.. 9256 00.. 9257 00.. 9258 00.. 9259 00.. 9260 00.. 9261 00.. 9262 00.. 9263 00.. 9264 00.. Page 3 MS 16 - suppl mat - file_s5.txt 9265 00.. 9266 00.. 9267 00.. 9268 00.. 9269 00.. 9270 00.. 9271 00.. 9272 00..

Page 4 MS 16 - suppl mat - file_s4.txt PEDINFO -- 14 Jan 2014 10:52:21 -- [S.A.G.E. v6.2.0; 20 Jan 2012] Remember you have agreed to add an appropriate statement (including the NIH grant number) under "acknowledgments" in any publication of results obtained by using this program. Suggested wording is: "(Some of)The results of this paper were obtained by using the software package S.A.G.E., which was supported by a U.S. Public Health Service Resource Grant (RR03655) from the National Center for Research Resources." ======| General Statistics: All Pedigrees | |======| | | Count| Mean Size +/- Std. Dev. ( Min., Max.) | |------| |Pedigrees | 1| 196.00 +/- --- ( 196, 196) | |======| |Generation Statistics|| Nuc Family Statistics || Inh Vector Bit Stats | |# of Gens | # of Peds|| # of Nuc Fams |# of Peds|| # of Bits |# of Peds | |------| | undet.| 1|| 65 - 128| 1|| 129 - 256| 1| |======| | | Count| Mean Size +/- Std. Dev. ( Min., Max.) | |------| |Sibships | 67| 2.01 +/- 2.11 ( 1, 12) | |======| |Constituent | || Marriage | || | | |Pedigrees | 1|| Rings | 0|| Loops | 11| |======| |Pairs | Count|| Individuals | Count| |------| |Parent/Off | 270|| Male | 94| |Sib/Sib | 218|| Female | 101| |Sis/Sis | 75|| Unknown | 0| |Bro/Bro | 32|| Total | 196| |Bro/Sis | 111|| | | |Grandp. | 318|| Founder | 57| |Avunc. | 438|| Non-founder | 135| |Half Sib | 12|| Singleton | 4| |Cousin | 912|| Total | 196| |======| | Individuals With Multiple Mates | |(Pedigree, Individual) Mates | |------| |(aic_gemepo_v3, 1231) 2320, 2511 | |(aic_gemepo_v3, 59) 68, 57 | |(aic_gemepo_v3, 61) 1183, 9999 | |(aic_gemepo_v3, 7682) 7683, 7684 | |======| | Consanguineous Mating Pairs | |Pedigree Pair | |------| |aic_gemepo_v3 1007, 771 | |aic_gemepo_v3 1027, 763 | |aic_gemepo_v3 1071, 2506 | |aic_gemepo_v3 1073, 2307 | |aic_gemepo_v3 1077, 2443 | |aic_gemepo_v3 1211, 901 | |aic_gemepo_v3 1714, 1835 | |aic_gemepo_v3 1842, 2908 | |aic_gemepo_v3 2261, 2622 | |aic_gemepo_v3 2295, 63 | |aic_gemepo_v3 59, 68 | Page 1 MS 16 - suppl mat - file_s4.txt ======

======| Trait Statistics: All Pedigrees, Trait - ALBINO | |======| | | 0 Parents w. Data| 1 Parent w. Data| 2 Parents w. Data| |------| |Sibships | 0| 28| 39| |======| | | Count| Mean Size +/- Std. Dev. ( Min., Max.) | |------| |Sibships | 67| 2.01 +/- 2.11 ( 1, 12) | |------| |Pedigrees | 1| 164.00 +/- --- ( 164, 164) | |======| | Nuc Family Statistics | | # of Nuc Fams |# of Peds | |------| | 65 - 128| 1 | |======| |Pairs | Concord.| Discord.| Concord.| Uninform.| Total| Corr.| | | Unaff.| | Aff.| | | | |------| |Parent/Off| 201| 29| 0| 40| 270| --- | |Sib/Sib | 204| 0| 14| 0| 218| 1.0000| |Sis/Sis | 66| 0| 9| 0| 75| 1.0000| |Bro/Bro | 29| 0| 3| 0| 32| 1.0000| |Bro/Sis | 109| 0| 2| 0| 111| 1.0000| |Grandp. | 228| 40| 0| 50| 318| --- | |Avunc. | 438| 0| 0| 0| 438| --- | |Half Sib | 12| 0| 0| 0| 12| --- | |Cousin | 912| 0| 0| 0| 912| --- | |======| |Indiv.'s | Affected| Unaff.| Missing| Total| | | |------| |Male | 9| 71| 14| 94| | | |Female | 10| 75| 17| 102| | | |Unknown | 0| 0| 0| 0| | | | Total | 19| 145| 31| 196| | | | | | | | | | | |Founder | 0| 29| 28| 57| | | |Nonfound. | 19| 116| 0| 135| | | |Singleton | 0| 0| 3| 3| | | | Total | 19| 145| 31| 195| | | ======

Page 2 Pedigree name indiv_idnum father_idnum mother_idnum Gender Affected aic_gemepo_v3 41 63 2295 F 1 aic_gemepo_v3 55 0 0 M 1 aic_gemepo_v3 56 0 0 M 1 aic_gemepo_v3 57 0 0 F 1 aic_gemepo_v3 58 0 0 F 1 aic_gemepo_v3 59 9244 74 M 1 aic_gemepo_v3 60 0 0 M 1 aic_gemepo_v3 61 73 75 F 1 aic_gemepo_v3 62 73 75 M 1 aic_gemepo_v3 63 73 75 M 1 aic_gemepo_v3 64 73 75 F 1 aic_gemepo_v3 65 73 75 F 1 aic_gemepo_v3 66 73 75 F 1 aic_gemepo_v3 67 73 75 M 1 aic_gemepo_v3 68 73 75 F 1 aic_gemepo_v3 69 76 1672 M 1 aic_gemepo_v3 70 76 1672 F 1 aic_gemepo_v3 71 69 1673 M 1 aic_gemepo_v3 72 69 1673 M 1 aic_gemepo_v3 73 0 0 M 1 aic_gemepo_v3 74 77 79 F 1 aic_gemepo_v3 75 77 79 F 1 aic_gemepo_v3 76 81 1671 M 1 aic_gemepo_v3 77 0 0 M 1 aic_gemepo_v3 78 82 83 M 1 aic_gemepo_v3 79 82 83 F 1 aic_gemepo_v3 80 82 83 F 1 aic_gemepo_v3 81 82 83 M 1 aic_gemepo_v3 82 0 0 M 1 aic_gemepo_v3 83 7681 9245 F 1 aic_gemepo_v3 553 62 58 M 1 aic_gemepo_v3 554 62 58 M 1 aic_gemepo_v3 555 62 58 M 1 aic_gemepo_v3 556 62 58 F 1 aic_gemepo_v3 557 62 58 F 1 aic_gemepo_v3 558 62 58 F 1 aic_gemepo_v3 559 62 58 F 1 aic_gemepo_v3 801 67 1683 F 1 aic_gemepo_v3 802 67 1683 F 1 aic_gemepo_v3 803 67 1683 F 1 aic_gemepo_v3 804 67 1683 F 1 aic_gemepo_v3 805 67 1683 M 1 aic_gemepo_v3 806 67 1683 M 1 aic_gemepo_v3 819 56 64 M 1 aic_gemepo_v3 820 56 64 M 1 aic_gemepo_v3 821 56 64 F 1 aic_gemepo_v3 822 56 64 F 1 aic_gemepo_v3 823 56 64 F 1 aic_gemepo_v3 826 56 64 F 1 aic_gemepo_v3 1017 60 66 M 1 aic_gemepo_v3 1018 60 66 F 1 aic_gemepo_v3 1019 60 66 F 1 aic_gemepo_v3 1183 0 0 M 1 aic_gemepo_v3 1189 1183 61 M 1 aic_gemepo_v3 1671 0 0 F 1 aic_gemepo_v3 1672 0 0 F 1 aic_gemepo_v3 1673 0 0 F 1 aic_gemepo_v3 1683 0 0 F 1 aic_gemepo_v3 2269 9999 61 F 1 aic_gemepo_v3 2279 55 65 M 1 aic_gemepo_v3 2280 55 65 F 1 aic_gemepo_v3 2281 55 65 M 1 aic_gemepo_v3 2290 59 68 M 1 aic_gemepo_v3 2291 59 68 F 1 aic_gemepo_v3 2292 59 68 F 1 aic_gemepo_v3 2293 59 57 F 1 aic_gemepo_v3 2294 59 57 F 1 aic_gemepo_v3 2295 59 57 F 1 aic_gemepo_v3 2306 63 2295 M 1 aic_gemepo_v3 2307 63 2295 M 1 aic_gemepo_v3 2308 63 2295 M 1 aic_gemepo_v3 2309 63 2295 F 1 aic_gemepo_v3 2310 63 2295 F 1 aic_gemepo_v3 2311 63 2295 F 1 aic_gemepo_v3 2312 63 2295 F 1 aic_gemepo_v3 2313 63 2295 F 1 aic_gemepo_v3 2314 63 2295 M 1 aic_gemepo_v3 3375 67 1683 F 1 aic_gemepo_v3 3382 67 1683 F 1 aic_gemepo_v3 3383 67 1683 M 1 aic_gemepo_v3 3384 67 1683 F 1 aic_gemepo_v3 3385 67 1683 M 1 aic_gemepo_v3 3386 67 1683 M 1 aic_gemepo_v3 7681 7682 7683 M 1 aic_gemepo_v3 7682 0 0 M 1 aic_gemepo_v3 7683 0 0 F 1 aic_gemepo_v3 7684 0 0 F 1 aic_gemepo_v3 7685 7682 7684 M 1 aic_gemepo_v3 7686 0 0 F 1 aic_gemepo_v3 7687 7685 7686 F 1 aic_gemepo_v3 7688 0 0 M 1 aic_gemepo_v3 7689 7688 7687 M 1 aic_gemepo_v3 7690 0 0 F 1 aic_gemepo_v3 7691 7689 7690 M 1 aic_gemepo_v3 7692 0 0 F 1 aic_gemepo_v3 7694 7691 7692 F 1 aic_gemepo_v3 7695 0 0 M 1 aic_gemepo_v3 7698 7695 7696 M 1 aic_gemepo_v3 7699 0 0 F 1 aic_gemepo_v3 7700 7698 7694 M 1 aic_gemepo_v3 9999 0 0 M 1 aic_gemepo_v3 7696 0 0 F 1 aic_gemepo_v3 9109 0 0 M aic_gemepo_v3 9134 9135 0 M aic_gemepo_v3 9135 0 0 M aic_gemepo_v3 9138 0 0 M aic_gemepo_v3 167 71 161 M 1 aic_gemepo_v3 183 167 9246 M 1 aic_gemepo_v3 185 183 9247 M 1 aic_gemepo_v3 525 185 9248 F 2 aic_gemepo_v3 527 185 9248 F 2 aic_gemepo_v3 529 185 9248 F 2 aic_gemepo_v3 161 0 0 F 1 aic_gemepo_v3 1227 1648 70 M 1 aic_gemepo_v3 1667 1227 9249 M 1 aic_gemepo_v3 1225 1227 9249 F 1 aic_gemepo_v3 1224 1227 9249 F 1 aic_gemepo_v3 2321 1667 9250 M 1 aic_gemepo_v3 1111 2321 9251 M 1 aic_gemepo_v3 1714 1111 9252 M 1 aic_gemepo_v3 1842 1714 1835 M 1 aic_gemepo_v3 2920 1842 2908 M 2 aic_gemepo_v3 2921 1842 2908 M 2 aic_gemepo_v3 35 9256 1224 M 1 aic_gemepo_v3 1231 9256 1224 M 1 aic_gemepo_v3 1235 9256 1224 M 1 aic_gemepo_v3 2886 35 9257 F 1 aic_gemepo_v3 2908 9258 2886 F 1 aic_gemepo_v3 1835 1235 566 F 1 aic_gemepo_v3 566 0 0 F 1 aic_gemepo_v3 2325 1231 2320 M 1 aic_gemepo_v3 2 2325 9259 M 2 aic_gemepo_v3 1419 2325 9259 M 2 aic_gemepo_v3 2320 0 0 F 1 aic_gemepo_v3 2511 0 0 F 1 aic_gemepo_v3 2619 1231 2511 F 1 aic_gemepo_v3 2622 9260 2619 F 1 aic_gemepo_v3 1901 9254 1225 M 1 aic_gemepo_v3 2261 1901 9255 M 1 aic_gemepo_v3 2646 2261 2622 M 2 aic_gemepo_v3 2648 2261 2622 M 2 aic_gemepo_v3 1648 0 0 M 1 aic_gemepo_v3 899 9263 822 F 1 aic_gemepo_v3 901 9264 899 M 1 aic_gemepo_v3 1399 901 1211 M 2 aic_gemepo_v3 1394 901 1211 F 2 aic_gemepo_v3 1191 1189 9262 M 1 aic_gemepo_v3 1211 1191 9261 F 1 aic_gemepo_v3 1005 9266 826 M 1 aic_gemepo_v3 1007 1005 9265 M 1 aic_gemepo_v3 800 1007 771 F 2 aic_gemepo_v3 763 9267 2310 M 1 aic_gemepo_v3 771 763 1027 F 1 aic_gemepo_v3 1027 1017 9268 F 1 aic_gemepo_v3 1073 9269 1018 F 1 aic_gemepo_v3 1077 2307 1073 M 1 aic_gemepo_v3 2317 9270 2309 M 1 aic_gemepo_v3 2443 2317 9271 F 1 aic_gemepo_v3 2446 1077 2443 F 2 aic_gemepo_v3 2447 1077 2443 M 2 aic_gemepo_v3 1071 9269 1018 M 1 aic_gemepo_v3 2506 9267 2310 F 1 aic_gemepo_v3 2507 1071 2506 M 1 aic_gemepo_v3 1451 2507 9272 F 2 aic_gemepo_v3 1454 2507 9272 F 2 aic_gemepo_v3 1458 2507 9272 F 2 aic_gemepo_v3 1459 2507 9272 F 2 aic_gemepo_v3 7701 7700 7699 M 2 aic_gemepo_v3 9244 0 0 M aic_gemepo_v3 9245 0 0 F aic_gemepo_v3 9246 0 0 F aic_gemepo_v3 9247 0 0 F aic_gemepo_v3 9248 0 0 F aic_gemepo_v3 9249 0 0 F aic_gemepo_v3 9250 0 0 F aic_gemepo_v3 9251 0 0 F aic_gemepo_v3 9252 0 0 F aic_gemepo_v3 9254 0 0 M aic_gemepo_v3 9255 0 0 F aic_gemepo_v3 9256 0 0 M aic_gemepo_v3 9257 0 0 F aic_gemepo_v3 9258 0 0 M aic_gemepo_v3 9259 0 0 F aic_gemepo_v3 9260 0 0 M aic_gemepo_v3 9261 0 0 F aic_gemepo_v3 9262 0 0 F aic_gemepo_v3 9263 0 0 M aic_gemepo_v3 9264 0 0 M aic_gemepo_v3 9265 0 0 F aic_gemepo_v3 9266 0 0 M aic_gemepo_v3 9267 0 0 M aic_gemepo_v3 9268 0 0 F aic_gemepo_v3 9269 0 0 M aic_gemepo_v3 9270 0 0 M aic_gemepo_v3 9271 0 0 F aic_gemepo_v3 9272 0 0 F MS 16 - suppl mat - file_s2.txt id fid mid sex aff 2 2325 9259 1 2 35 9256 1224 1 1 41 63 2295 2 1 550011 560011 570021 580021 59 9244 74 1 1 600011 61 73 75 2 1 62 73 75 1 1 63 73 75 1 1 64 73 75 2 1 65 73 75 2 1 66 73 75 2 1 67 73 75 1 1 68 73 75 2 1 69 76 1672 1 1 70 76 1672 2 1 71 69 1673 1 1 72 69 1673 1 1 730011 74 77 79 2 1 75 77 79 2 1 76 81 1671 1 1 770011 78 82 83 1 1 79 82 83 2 1 80 82 83 2 1 81 82 83 1 1 820011 83 7681 9245 2 1 1610021 167 71 161 1 1 183 167 9246 1 1 185 183 9247 1 1 525 185 9248 2 2 527 185 9248 2 2 529 185 9248 2 2 553 62 58 1 1 554 62 58 1 1 555 62 58 1 1 556 62 58 2 1 557 62 58 2 1 558 62 58 2 1 559 62 58 2 1 5660021 763 9267 2310 1 1 771 763 1027 2 1 800 1007 771 2 2 801 67 1683 2 1 802 67 1683 2 1 803 67 1683 2 1 804 67 1683 2 1 805 67 1683 1 1 806 67 1683 1 1 819 56 64 1 1 820 56 64 1 1 821 56 64 2 1 822 56 64 2 1 823 56 64 2 1 826 56 64 2 1 Page 1 MS 16 - suppl mat - file_s2.txt 899 9263 822 2 1 901 9264 899 1 1 1005 9266 826 1 1 1007 1005 9265 1 1 1017 60 66 1 1 1018 60 66 2 1 1019 60 66 2 1 1027 1017 9268 2 1 1071 9269 1018 1 1 1073 9269 1018 2 1 1077 2307 1073 1 1 1111 2321 9251 1 1 1183 0011 1189 1183 61 1 1 1191 1189 9262 1 1 1211 1191 9261 2 1 1224 1227 9249 2 1 1225 1227 9249 2 1 1227 1648 70 1 1 1231 9256 1224 1 1 1235 9256 1224 1 1 1394 901 1211 2 2 1399 901 1211 1 2 1419 2325 9259 1 2 1451 2507 9272 2 2 1454 2507 9272 2 2 1458 2507 9272 2 2 1459 2507 9272 2 2 1648 0011 1667 1227 9249 1 1 1671 0021 1672 0021 1673 0021 1683 0021 1714 1111 9252 1 1 1835 1235 566 2 1 1842 1714 1835 1 1 1901 9254 1225 1 1 2261 1901 9255 1 1 2269 9999 61 2 1 2279 55 65 1 1 2280 55 65 2 1 2281 55 65 1 1 2290 59 68 1 1 2291 59 68 2 1 2292 59 68 2 1 2293 59 57 2 1 2294 59 57 2 1 2295 59 57 2 1 2306 63 2295 1 1 2307 63 2295 1 1 2308 63 2295 1 1 2309 63 2295 2 1 2310 63 2295 2 1 2311 63 2295 2 1 2312 63 2295 2 1 2313 63 2295 2 1 2314 63 2295 1 1 2317 9270 2309 1 1 2320 0021 2321 1667 9250 1 1 2325 1231 2320 1 1 2443 2317 9271 2 1 Page 2 MS 16 - suppl mat - file_s2.txt 2446 1077 2443 2 2 2447 1077 2443 1 2 2506 9267 2310 2 1 2507 1071 2506 1 1 2511 0021 2619 1231 2511 2 1 2622 9260 2619 2 1 2646 2261 2622 1 2 2648 2261 2622 1 2 2886 35 9257 2 1 2908 9258 2886 2 1 2920 1842 2908 1 2 2921 1842 2908 1 2 3375 67 1683 2 1 3382 67 1683 2 1 3383 67 1683 1 1 3384 67 1683 2 1 3385 67 1683 1 1 3386 67 1683 1 1 7681 7682 7683 1 1 7682 0011 7683 0021 7684 0021 7685 7682 7684 1 1 7686 0021 7687 7685 7686 2 1 7688 0011 7689 7688 7687 1 1 7690 0021 7691 7689 7690 1 1 7692 0021 7694 7691 7692 2 1 7695 0011 7696 0021 7698 7695 7696 1 1 7699 0021 7700 7698 7694 1 1 7701 7700 7699 1 2 9109 0 0 1 9134 9135 9273 1 9135 0 0 1 9138 0 0 1 9244 0 0 1 9245 0 0 2 9246 0 0 2 9247 0 0 2 9248 0 0 2 9249 0 0 2 9250 0 0 2 9251 0 0 2 9252 0 0 2 9254 0 0 1 9255 0 0 2 9256 0 0 1 9257 0 0 2 9258 0 0 1 9259 0 0 2 9260 0 0 1 9261 0 0 2 9262 0 0 2 9263 0 0 1 9264 0 0 1 9265 0 0 2 Page 3 MS 16 - suppl mat - file_s2.txt 9266 0 0 1 9267 0 0 1 9268 0 0 2 9269 0 0 1 9270 0 0 1 9271 0 0 2 9272 0 0 2 9273 0 0 2 9999 0011

Page 4 key Pedigree name indiv_idnum father_idnum mother_idnum Gender Affected aic_41 aic_subped2 41 63 2295 F 1 aic_55 aic_subped2 55 0 0 M 1 aic_56 aic_subped2 56 0 0 M 1 aic_57 aic_subped2 57 0 0 F 1 aic_58 aic_subped2 58 0 0 F 1 aic_59 aic_subped2 59 9244 74 M 1 aic_60 aic_subped2 60 0 0 M 1 aic_61 aic_subped2 61 73 75 F 1 aic_62 aic_subped2 62 73 75 M 1 aic_63 aic_subped2 63 73 75 M 1 aic_64 aic_subped2 64 73 75 F 1 aic_65 aic_subped2 65 73 75 F 1 aic_66 aic_subped2 66 73 75 F 1 aic_67 aic_subped2 67 73 75 M 1 aic_68 aic_subped2 68 73 75 F 1 aic_69 aic_subped2 69 76 1672 M 1 aic_70 aic_subped2 70 76 1672 F 1 aic_71 aic_subped2 71 69 1673 M 1 aic_72 aic_subped2 72 69 1673 M 1 aic_73 aic_subped2 73 0 0 M 1 aic_74 aic_subped2 74 77 79 F 1 aic_75 aic_subped2 75 77 79 F 1 aic_76 aic_subped2 76 81 1671 M 1 aic_77 aic_subped2 77 0 0 M 1 aic_78 aic_subped2 78 82 83 M 1 aic_79 aic_subped2 79 82 83 F 1 aic_80 aic_subped2 80 82 83 F 1 aic_81 aic_subped2 81 82 83 M 1 aic_82 aic_subped2 82 0 0 M 1 aic_83 aic_subped2 83 7681 9245 F 1 aic_553 aic_subped2 553 62 58 M 1 aic_554 aic_subped2 554 62 58 M 1 aic_555 aic_subped2 555 62 58 M 1 aic_556 aic_subped2 556 62 58 F 1 aic_557 aic_subped2 557 62 58 F 1 aic_558 aic_subped2 558 62 58 F 1 aic_559 aic_subped2 559 62 58 F 1 aic_801 aic_subped2 801 67 1683 F 1 aic_802 aic_subped2 802 67 1683 F 1 aic_803 aic_subped2 803 67 1683 F 1 aic_804 aic_subped2 804 67 1683 F 1 aic_805 aic_subped2 805 67 1683 M 1 aic_806 aic_subped2 806 67 1683 M 1 aic_819 aic_subped2 819 56 64 M 1 aic_820 aic_subped2 820 56 64 M 1 aic_821 aic_subped2 821 56 64 F 1 aic_822 aic_subped2 822 56 64 F 1 aic_823 aic_subped2 823 56 64 F 1 aic_826 aic_subped2 826 56 64 F 1 aic_1017 aic_subped2 1017 60 66 M 1 aic_1018 aic_subped2 1018 60 66 F 1 aic_1019 aic_subped2 1019 60 66 F 1 aic_1183 aic_subped2 1183 0 0 M 1 aic_1189 aic_subped2 1189 1183 61 M 1 aic_1671 aic_subped2 1671 0 0 F 1 aic_1672 aic_subped2 1672 0 0 F 1 aic_1673 aic_subped2 1673 0 0 F 1 aic_1683 aic_subped2 1683 0 0 F 1 aic_2269 aic_subped2 2269 9999 61 F 1 aic_2279 aic_subped2 2279 55 65 M 1 aic_2280 aic_subped2 2280 55 65 F 1 aic_2281 aic_subped2 2281 55 65 M 1 aic_2290 aic_subped2 2290 59 68 M 1 aic_2291 aic_subped2 2291 59 68 F 1 aic_2292 aic_subped2 2292 59 68 F 1 aic_2293 aic_subped2 2293 59 57 F 1 aic_2294 aic_subped2 2294 59 57 F 1 aic_2295 aic_subped2 2295 59 57 F 1 aic_2306 aic_subped2 2306 63 2295 M 1 aic_2307 aic_subped2 2307 63 2295 M 1 aic_2308 aic_subped2 2308 63 2295 M 1 aic_2309 aic_subped2 2309 63 2295 F 1 aic_2310 aic_subped2 2310 63 2295 F 1 aic_2311 aic_subped2 2311 63 2295 F 1 aic_2312 aic_subped2 2312 63 2295 F 1 aic_2313 aic_subped2 2313 63 2295 F 1 aic_2314 aic_subped2 2314 63 2295 M 1 aic_3375 aic_subped2 3375 67 1683 F 1 aic_3382 aic_subped2 3382 67 1683 F 1 aic_3383 aic_subped2 3383 67 1683 M 1 aic_3384 aic_subped2 3384 67 1683 F 1 aic_3385 aic_subped2 3385 67 1683 M 1 aic_3386 aic_subped2 3386 67 1683 M 1 aic_7681 aic_subped2 7681 7682 7683 M 1 aic_7682 aic_subped2 7682 0 0 M 1 aic_7683 aic_subped2 7683 0 0 F 1 aic_7684 aic_subped2 7684 0 0 F 1 aic_7685 aic_subped2 7685 7682 7684 M 1 aic_7686 aic_subped2 7686 0 0 F 1 aic_7687 aic_subped2 7687 7685 7686 F 1 aic_7688 aic_subped2 7688 0 0 M 1 aic_7689 aic_subped2 7689 7688 7687 M 1 aic_7690 aic_subped2 7690 0 0 F 1 aic_7691 aic_subped2 7691 7689 7690 M 1 aic_7692 aic_subped2 7692 0 0 F 1 aic_7694 aic_subped2 7694 7691 7692 F 1 aic_7695 aic_subped2 7695 0 0 M 1 aic_7698 aic_subped2 7698 7695 7696 M 1 aic_7699 aic_subped2 7699 0 0 F 1 aic_7700 aic_subped2 7700 7698 7694 M 1 aic_9999 aic_subped2 9999 0 0 M 1 aic_7696 aic_subped2 7696 0 0 F 1 aic_9109 aic_subped2 9109 0 0 M . aic_9134 aic_subped2 9134 9135 0 M . aic_9135 aic_subped2 9135 0 0 M . aic_9138 aic_subped2 9138 0 0 M . aic_167 aic_subped2 167 71 161 M 1 aic_183 aic_subped2 183 167 9246 M 1 aic_185 aic_subped2 185 183 9247 M 1 aic_525 aic_subped2 525 185 9248 F 2 aic_527 aic_subped2 527 185 9248 F 2 aic_529 aic_subped2 529 185 9248 F 2 aic_161 aic_subped2 161 0 0 F 1 aic_1227 aic_subped2 1227 1648 70 M 1 aic_1667 aic_subped2 1667 1227 9249 M 1 aic_1225 aic_subped2 1225 1227 9249 F 1 aic_1224 aic_subped2 1224 1227 9249 F 1 aic_2321 aic_subped2 2321 1667 9250 M 1 aic_1111 aic_subped2 1111 2321 9251 M 1 aic_1714 aic_subped2 1714 1111 9252 M 1 aic_1842 aic_subped2 1842 1714 1835 M 1 aic_2920 aic_subped2 2920 1842 2908 M 2 aic_2921 aic_subped2 2921 1842 2908 M 2 aic_35 aic_subped2 35 9256 1224 M 1 aic_1231 aic_subped2 1231 9256 1224 M 1 aic_1235 aic_subped2 1235 9256 1224 M 1 aic_2886 aic_subped2 2886 35 9257 F 1 aic_2908 aic_subped2 2908 9258 2886 F 1 aic_1835 aic_subped2 1835 1235 566 F 1 aic_566 aic_subped2 566 0 0 F 1 aic_2325 aic_subped2 2325 1231 2320 M 1 aic_2 aic_subped2 2 2325 9259 M 2 aic_1419 aic_subped2 1419 2325 9259 M 2 aic_2320 aic_subped2 2320 0 0 F 1 aic_2511 aic_subped2 2511 0 0 F 1 aic_2619 aic_subped2 2619 1231 2511 F 1 aic_2622 aic_subped2 2622 9260 2619 F 1 aic_1901 aic_subped2 1901 9254 1225 M 1 aic_2261 aic_subped2 2261 1901 9255 M 1 aic_2646 aic_subped2 2646 2261 2622 M 2 aic_2648 aic_subped2 2648 2261 2622 M 2 aic_1648 aic_subped2 1648 0 0 M 1 aic_899 aic_subped2 899 9263 822 F 1 aic_901 aic_subped2 901 9264 899 M 1 aic_1399 aic_subped2 1399 901 1211 M 2 aic_1394 aic_subped2 1394 901 1211 F 2 aic_1191 aic_subped2 1191 1189 9262 M 1 aic_1211 aic_subped2 1211 1191 9261 F 1 aic_1005 aic_subped2 1005 9266 826 M 1 aic_1007 aic_subped2 1007 1005 9265 M 1 aic_800 aic_subped2 800 1007 771 F 2 aic_763 aic_subped2 763 9267 2310 M 1 aic_771 aic_subped2 771 763 1027 F 1 aic_1027 aic_subped2 1027 1017 9268 F 1 aic_1073 aic_subped2 1073 9269 1018 F 1 aic_1077 aic_subped2 1077 2307 1073 M 1 aic_2317 aic_subped2 2317 9270 2309 M 1 aic_2443 aic_subped2 2443 2317 9271 F 1 aic_2446 aic_subped2 2446 1077 2443 F 2 aic_2447 aic_subped2 2447 1077 2443 M 2 aic_1071 aic_subped2 1071 9269 1018 M 1 aic_2506 aic_subped2 2506 9267 2310 F 1 aic_2507 aic_subped2 2507 1071 2506 M 1 aic_1451 aic_subped2 1451 2507 9272 F 2 aic_1454 aic_subped2 1454 2507 9272 F 2 aic_1458 aic_subped2 1458 2507 9272 F 2 aic_1459 aic_subped2 1459 2507 9272 F 2 aic_7701 aic_subped2 7701 7700 7699 M 2 aic_9244 aic_subped2 9244 0 0 M . aic_9245 aic_subped2 9245 0 0 F . aic_9246 aic_subped2 9246 0 0 F . aic_9247 aic_subped2 9247 0 0 F . aic_9248 aic_subped2 9248 0 0 F . aic_9249 aic_subped2 9249 0 0 F . aic_9250 aic_subped2 9250 0 0 F . aic_9251 aic_subped2 9251 0 0 F . aic_9252 aic_subped2 9252 0 0 F . aic_9254 aic_subped2 9254 0 0 M . aic_9255 aic_subped2 9255 0 0 F . aic_9256 aic_subped2 9256 0 0 M . aic_9257 aic_subped2 9257 0 0 F . aic_9258 aic_subped2 9258 0 0 M . aic_9259 aic_subped2 9259 0 0 F . aic_9260 aic_subped2 9260 0 0 M . aic_9261 aic_subped2 9261 0 0 F . aic_9262 aic_subped2 9262 0 0 F . aic_9263 aic_subped2 9263 0 0 M . aic_9264 aic_subped2 9264 0 0 M . aic_9265 aic_subped2 9265 0 0 F . aic_9266 aic_subped2 9266 0 0 M . aic_9267 aic_subped2 9267 0 0 M . aic_9268 aic_subped2 9268 0 0 F . aic_9269 aic_subped2 9269 0 0 M . aic_9270 aic_subped2 9270 0 0 M . aic_9271 aic_subped2 9271 0 0 F . aic_9272 aic_subped2 9272 0 0 F .